Use of geldanamycin and related compounds for prophylaxis or treatment of fibrogenic disorders

ABSTRACT

A method for prophylaxis or treatment of a mammal, particularly human, at risk for a fibrogenic disorder is disclosed. The compositions and methods of the invention are directed both to treatments for existing fibrogenic disorders and prevention thereof. Such disorders include, but are not limited to, connective tissue diseases, such as scleroderma (or systemic sclerosis), polymyositis, systemic lupus erythematosis and rheumatoid arthristis, and other fibrotic disorders, including liver cirrhosis, keloid formation, interstitial nephritis and pulmonary fibrosis. A therapeutic composition according to the invention includes, as a therapeutic agent, an inhibitor of a collagen promoter in a pharmaceutically acceptable inert carrier vehicle, preferably for local, and particularly topical, application. Exemplary inhibitors include those that interfere with heat shock protein 90 (Hsp 90) chaperone function, e.g., the specific inhibitor geldanamycin or other known Hsp90 inhibitors such as macbecin I and II, herbimycin, radcicol and novobiocin.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. ProvisionalApplication No. 60/214,950 filed Jun. 29, 2000 entitled, A USE FORGELDANAMYCIN AND DERIVATIVES AS INHIBITORS OF TGF-β SIGNALING ANDEXTRACELLULAR MATRIX SYNTHESIS, the whole of which is herebyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Part of the work leading to this invention was carried out withUnited States Government support provided by the National Institutes ofHealth under Grant Nos. RO1 AR-32343 and P60 AR-20613. Therefore, theU.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Systemic sclerosis, or scleroderma (SSc), is characterized bythree features: increased extracellular matrix accumulation in the skinand organs, vascular injury and tissue ischemia, and immune cellactivation and autoantibody production. The disease is one of a class ofconnective tissue diseases including polymyositis, systemic lupuserythematosis, and rheumatoid arthritis, which all share some commonimmunological features and are sometimes found within families.

[0004] Several studies show specific HLA patterns associated withautoantibody production (1). However, family studies in the generalpopulation show only a weak genetic predisposition to scleroderma. Oneexception to this has been described for Choctaw Native Americans (2).This group has a high prevalence of scleroderma (469/100,000) and an HLADR2 haplotype that is strongly associated with the disease. Preliminarymapping suggests that there may be a defect in the fibrillin gene (3),which is also thought to be defective in one of the few animal modelsfor scleroderma, that of the mutant mouse line, Tsk-1 (4,5).

[0005] While the disease may have an underlying genetic basis in somecases, in the general population it is a complex trait that involvesgenetic risk factors and, in some cases, may also involve environmentaltoxins (6,7). New methods of treating scleroderma and related conditionswould be very useful.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention encompasses both prophylactic andtherapeutic treatments for a mammal, preferably a human, at risk for afibrogenic disorder. In particular, the compositions and methods of thepresent invention are directed both to treatments for existingfibrogenic disorders and prevention thereof. Such disorders include, butare not limited to, connective tissue diseases, such as scleroderma (orsystemic sclerosis), polymyositis, systemic lupus erythematosis andrheumatoid arthritis, and other fibrotic disorders, including livercirrhosis, keloid formation, interstitial nephritis and pulmonaryfibrosis.

[0007] A therapeutic composition according to the invention includes, asa therapeutic agent, an inhibitor of collagen promoter activity in apharmaceutically acceptable inert carrier vehicle, preferably for local,and particularly topical, application. Exemplary inhibitors includethose that interfere with heat shock protein 90 (Hsp90) chaperonefunction, e.g., the specific inhibitor geldanamycin or other known Hsp90inhibitors such as macbecin I and II, herbimycin, radcicol andnovobiocin.

[0008] In another embodiment, the present invention is directed to anarticle of manufacture comprising a packaging material and a therapeuticcomposition of the present invention contained within the packagingmaterial. The therapeutic composition is therapeutically effective forprophylaxis or treatment of fibrogenic disorders. The packaging materialalso comprises a label with instructions for use, which indicate thatthe therapeutic composition can be used for prophylaxis or treatment offibrogenic disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof andfrom the claims, taken in conjunction with the accompanying drawings, inwhich:

[0010]FIG. 1A shows hybridization of labeled mRNA from culturedscleroderma fibroblasts to a large array of expressed sequence tags.FIG. 1B shows hybridization of mRNA from 3 healthy fibroblast lines(N-96-05, N-96-02, N-92-04) and from 3 scleroderma lines (96-02-A,97-03-A, 97-02-A) to identical large array filters. Each clone on thefilters is represented by two spots. R points to two reference cloneswhose intensity is unchanged between the healthy and scleroderma cellswhen compared to spots in other regions of the filter. A, B and C arecomparable to R in the healthy lines. However, A, B and C areseveral-fold more intense than R in the scleroderma lines;

[0011]FIG. 2 shows northern analysis using the insert from clone A ofFIG. 1B as a probe. Total RNA from lesional (L) or nonlesional (N)fibroblast lines from three scleroderma patients (P1-P3) or from 6healthy fibroblast lines (N1-N6) was analyzed. The arrow shows the sizeof the Hsp90 message;

[0012]FIG. 3 shows use of a mouse monoclonal anti-Hsp90 antibody tovisualize Hsp90 protein in three healthy fibroblast lines (A-C) or inthree scleroderma fibroblast lines (D-F);

[0013]FIG. 4 shows human dermal fibroblasts (healthy and scleroderma)plated in equal numbers and cultured overnight. Where indicated, cellswere heat shocked at 42° for 1.5 hours. Nuclear extracts were preparedand bound to labeled duplex oligos as described in ExperimentalProcedures. Protein concentrations were determined for each sample andequal amounts of protein was bound and loaded onto the gel;

[0014]FIG. 5 shows NIH 3T3 fibroblasts co-transfected with an 804 bpproximal region of the collagen 1 α (I) promoter driving luciferase andeither an empty control vector (pBKRSV), an Hsp70 overexpressionplasmid, an Hsp90-α overexpression plasmid or an Hsp90-β overexpressionplasmid. Transfections were performed in triplicate and each extract wasassayed in duplicate. Extracts were harvested and normalized to theprotein concentration in each extract;

[0015]FIG. 6 shows NIH 3T3 fibroblasts co-transfected with a 4.7 kbregion of the human collagenase (MMP1) promoter driving a luciferasereporter and either an empty control vector (pBKRSV) or an Hsp90-βoverexpression plasmid. Transfections were performed in triplicate andeach extract was assayed in duplicate. Extracts were harvested andnormalized to the protein concentration in each extract;

[0016] FIGS. 7A-7C show fibroblasts subjected to 24-hour treatments withgeldanamycin, TGF or both. After 24 hours, total RNA was harvested andsubjected to northern analysis using an α1(I) collagen probe. (A)Northern analysis using NIH 3T3 fibroblasts. (B) Northern analysis usingprimary healthy human dermal fibroblasts. (C) Methylene blue staining ofthe filter in (B). Numbers represent relative intensities of the lowercollagen band after normalizing bands to GAPDH band intensity;

[0017]FIG. 8 shows NIH 3T3 fibroblasts transfected with either theTGF-sensitive reporter p3TPlux or pPN1-4.5. The latter contains a 4.5 kbfragment of the PN1 promoter driving the luciferase gene. Cells wereincubated for 24 hours after transfection, washed 3 times with PBS andthen subjected to a 24 hour treatment with geldanamycin, TGF or both, asindicated. Cells were then harvested and luciferase activitiesdetermined;

[0018]FIG. 9 shows Hsp90 stimulating the expression of 3TP lux intransiently transfected transgenic mice. Mice were injected in the tailvein with either the 3TPlux reporter and an Hsp90 overexpression vectoror the reporter and a pBKRSV empty vector control. Animal livers wereharvested at approximately 16 hours after injection and extracted inluciferase reporter buffer as described in Experimental Procedures. Eachbar represents the luciferase activity from a single mouse. All readingswere made in duplicate from a single liver extract. The actual datavalues for each injection are shown. No significant activity was foundin any animal injected with the control;

[0019]FIG. 10 shows NIH 3T3 cells transfected with either the pGL3promoter vector (containing a minimal promoter driving a luciferasereporter) or the pGL3 promoter vector containing a Smad binding element.The specific Hsp90 inhibitor, geldanamycin, was added four hours afterthe initiation of the transfection and replaced with fresh drug andmedium at 24 hours. Cells were harvested at 48 hours, proteinconcentrations of extracts determined and luciferase activity measured.Values shown are luciferase activities normalized to proteinconcentration; and

[0020] FIGS. 11A-11B show NIH 3T3 fibroblasts treated for 1 hour withthe indicated concentration of geldanamycin and then 15 minutes withTGF-β where indicated. (A) Cells were harvested and subjecting toelectrophoretic mobility shift analysis using a Smad binding element(SBE). Relative band intensities are shown. Where indicated, extractswere prepared with 100-fold higher levels of cold competitor probe. (B)Cells were subjected to 24 hour treatments with geldanamycin at variousconcentrations and extracts prepared and bound to control Oct-1-bindingduplex oligos to monitor non-specific effects of geldanamycin.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Fibroblasts grown from dermal scleroderma biopsies have beenshown to maintain a fibrogenic phenotype for several passages in culture(8,9). Since these cells maintain a disease-like phenotype, they are auseful model system for scleroderma skin and, therefore, are appropriatecandidates for analysis of differential gene expression.

[0022] In the work leading to the- invention, microarrays were utilizedto characterize the gene expression pattern in the sclerodermafibroblast model system. The goal of such experiments was tocharacterize individual genes from the scleroderma expressionfingerprint in order to identify those which might lie in a pathway ofexpressed genes responsible for the scleroderma phenotype—excessivematrix deposition. Initial differential display studies were expandedusing densely printed microarrays on nylon filter membranes.

[0023] In the current work, filter membranes containing large arrays ofexpressed sequence tags (ESTs) were probed with radioactively labeledmRNA isolated from independent scleroderma and healthy dermal fibroblastlines. These filters are commercially available and contain over 18,000different clones each spotted in duplicate. Four different sclerodermalesional, non-lesional and healthy lines—12 in all—were utilized assources of RNA. Several genes were consistently overexpressed orunderexpressed in the scleroderma lines compared to the healthycontrols. Among these was the gene for heat shock protein 90 alpha(Hsp90). Because of the 97% homology between the genes for Hsp90α andHsp90β, these genes were chosen for further study.

[0024] Hsp90 is a cytosolic protein conserved in species as diverse asbacteria and primates. One function of Hsp90 is to protect proteins fromaggregating during thermal stress (12). Another important function is asa hormone receptor chaperone. Genetic studies in yeast (13,14) andmolecular studies in higher cells (15) show that Hsp90 is required forhormone receptors to bind hormone. Without Hsp90, or in the presence ofthe specific inhibitor of Hsp90, the benzamycin ansiquinone geldanamycin(16), hormone receptor signal response fails.

[0025] The ATP binding site on Hsp90 (17) is required for normalchaperone function. Geldanamycin binds to the ATP binding site in Hsp90(18) and thus may block hormone receptor function by starving Hsp90 ofATP. Many different hormone receptors can bind Hsp90, including thosefor estrogen (19), progesterone (20), and aryl hydrocarbons (21)(including dioxin (22)). Hsp90 also exhibits chaperone activity forother signal transduction molecules, including casein kinase II (23),pp60^(v-src) (24), eIF2α kinase (25). Studies using null mutations ofHsp90 in Drosophila (Hsp83) show that it modulates Raf signaling (26)and signaling by the sevenless receptor tyrosine kinase (27).

[0026] Signal transduction by TGF-β may play an important role in thescleroderma phenotype. TGF-β is a potent fibrogenic cytokine thatstimulates the transcription and synthesis of multiple matrix-encodinggenes (28). The TGF-β family is thought to transduce its signal througha family of cytosolic proteins known as Smads (29). TGF-β binds to thetype II TGF-β receptor, which propagates the signal through the type Ireceptor to Smad. The family of human Smad proteins includes at least 9members (30). Smad 2 or Smad 3 proteins are transiently associated withthe type I receptor where they are activated by phosphorylation.Activated Smad2 or Smad 3 may bind to Smad 4 and be transported from thecytoplasm to the nucleus. Smad 3 and Smad 4 have been shown to bindspecific DNA sequences (31), including sequences in promoters that areactivated by TGF-β.

[0027] In the experiments described below, evidence that Hsp90 isoverexpressed in scleroderma fibroblasts is presented. It was shown,additionally, that Hsp90 overexpression induces the activity of acollagen reporter. Each of three different heat shock overexpressionconstructs caused a similar induction of collagen promoter activity.Since these three heat shock proteins are also part of a complex thatbinds the steroid hormone receptor, it is interesting that all threeinduce collagen. Also shown was that heat shock per se causes a similarinduction of endogenous collagen expression in 3T3 cells. In addition,Hsp90 overexpression reduced the transcription of a reporter driven bythe human MMP1 promoter. These effects show that Hsp90 overexpressionhas a physiological role in the accumulation of collagen in scleroderma,either by inducing its production or reducing its degradation.

[0028] An increased level of binding of heat shock factor 1 (HSF1) tothe consensus HSF1 binding site in scleroderma fibroblasts was alsofound. The HSF1 transcription factor is activated by a variety ofstresses, including heat, whereupon it forms a homotrimer polypeptideand migrates to the nucleus. In the nucleus, it binds to promoters ofseveral heat shock proteins and activates their transcription. Whetherincreased activity of this transcription factor is responsible for theincreased levels of Hsp90 mRNA in scleroderma fibroblasts is unknown,however, since other stress-induced transcription factors may play amore important role in Hsp90 transcription. Nonetheless, it isinteresting that scleroderma cells appear to activate HSF1 more readilythan healthy cells after the brief 1.5 hour heat shock treatment.

[0029] These experiments also show that Hsp90 overexpression orinhibition, respectively, enhances or blocks TGF-β signal transduction.This is relevant to the scleroderma condition since high TGF-β levelshave been found in the bronchial alveolar lavage fluid of sclerodermapatients with pulmonary fibrosis (32), and elevated levels of the TGF-βreceptor (both types I and II) have been described in sclerodermafibroblasts (33). Evidence is also provided here that Hsp90 acts on theTGF-β pathway by altering Smad function. Finally, through a combinationof a new method of mouse tail vein injection with anoverexpression/reporter-promoter system, in vivo evidence is generatedthat Hsp90 stimulates the TGF-β signal transduction pathway.

[0030] Experimental Procedures

[0031] Cell culture, RNA Isolation.

[0032] Patient biopsies, fibroblast culture and RNA isolation wereperformed as previously described (10). Biopsies were taken with patientconsent and the approval of the Institutional Review Board for HumanStudies at Boston University Medical Center. Lesional skin wasclinically identified and biopsies taken from the leading edge of thelesion, usually the forearm.

[0033] Hybridization to Large Array Filter Membranes.

[0034] Scleroderma or healthy fibroblast polyadenylated RNA was purifiedfrom 200-500 μg of total RNA using the polyA Spin mRNA Isolation Kitfrom New England Biolabs (Beverly, Mass.). Version 1.0 human EST filterarrays were purchased from Genome Systems, Inc. Hybridization andwashing using ³²P-labeled mRNA was performed in roller bottles followingthe manufacturer's recommendations with the following exceptions. Two μgof polyA RNA were labeled using avian reverse transcriptase (Promega,Madison, Wis.) at 42° instead of murine reverse transcriptase.Purification of incorporated from unincorporated counts was performed assuggested in the Genome Systems' protocol. The specific activity of theprobe was measured and the total amount of radioactivity in thehybridization adjusted to 1×10⁶ cpm per ml of hybridization fluid. Fiveμg of Cot1 human genomic DNA (Gibco BRL, Gaithersburg, Md.) was alsoincluded in the hybridization mixture.

[0035] Hybridization was performed overnight, and washed filters wereexposed to phosphoimage cassettes overnight (Molecular Dynamics, SanDiego, Calif.). Because the filters were too large for the cassettes,each filter was rotated 180° and exposed a second time. Thus, thecomplete filter was represented by two images.

[0036] Two filters were used 6 times each to image a total of 12 mRNAsamples. The filters were washed repeatedly using 15-minute soaks in 95°deionized water until the signal measured by a Geiger counter wasnegligible. The soak procedure was repeated 3-6 times to achievenegligible Geiger reading. The completeness of the washing procedure wasconfirmed by exposing washed filters to phosphoimage cassettesovernight.

[0037] The position of each spot on the filter is uniquely identified.With the position, the user can obtain an accession number and a partialsequence of the DNA clone. The manufacturer of the filter also suppliessamples of the clones that were spotted on the filter. Clone intensitieswere determined directly from the phosphoimages using DataMachinesoftware (34).

[0038] Sequencing and Northern Analysis.

[0039] The clone corresponding to Hsp90-α was purchased as an EST fromGenome Systems. The clone was originally misidentified by themanufacturer, but re-sequencing showed it to be Hsp90-α, andhybridization of the clone to the filter confirmed it was in fact Hsp90spotted at that position in the array. Sequencing was performed at theBoston University DNA/Protein Sequencing Core. Northern analysis ofHsp90 to total RNA from scleroderma and healthy fibroblasts wasperformed as previously described (10).

[0040] Immunocytochemistry.

[0041] A mouse monoclonal anti-Hsp90 antibody (AC88) was obtained fromStressGen (Victoria, BC). Scleroderma and healthy human dermalfibroblasts were applied to chamber slides using a Cytospin apparatusand fixed for 1 minute with 4% paraformaldehyde in PBS. The AC88antibody was diluted 1:500 in PBS with 5% dehydrated milk and incubatedin the washed, fixed chambers for 1 hour at room temperature. Sampleswere washed 4 times (5 minutes each) with PBS and a horseradishperoxidase-conjugated sheep anti-mouse secondary antibody (Amersham,Arlington Heights, Ill.; diluted 1:3000 in PBS) was added for 30 minutesat room temperature. Chambers were washed and enzyme activity visualizedusing TrueBlue (Kirkegaard and Perry Laboratories, Bethesda, Md.) andfollowing the manufacturer's protocol.

[0042] Transient transfection and Hsp constructs.

[0043] The complete open reading frame of Hsp70, corresponding toGenBank accession number M11717, was obtained from ATCC (Manassas, Va.);The complete open reading frame of Hsp90-α, corresponding to Riken #1127, was obtained from Riken Gene Bank with the permission of Dr.Kasunari Yokoyama (35). This is the full-length clone whose sequencematched that of the clone ‘A’ in FIG. 1B. The complete open readingframe of Hsp90-α, corresponding to GenBank accession number M16660, wasobtained from ATCC. Each of these three open reading frames was excisedand cloned into the expression pBKRSV (Stratagene, San Diego, Calif.).

[0044] Transient transfections were performed using lipofectamine (GibcoBRL) as follows: 10 μg (total) of indicated plasmid DNA was mixed with30 μl of lipofectamine and 500 μl of serum-free DMEM (Gibco). Thismixture was incubated at room temperature for 20 minutes. Ten cm dishesof sub-confluent 3T3 fibroblasts were rinsed with PBS, and 5 ml of serumfree DMEM added to each dish. The DNA/lipofectamine mixture was added toeach dish and incubated in the 37° CO₂ incubator for 4 hours. Ten ml ofDMEM with 15% serum was added and plates were incubated overnight.Medium was replaced with 10% serum at 24 hours and extracts of cellswere harvested at 48 hours. Cell extracts were obtained using PassiveLysis Buffer (Promega) and assays were performed according to thePromega protocol for pGL3 luciferase vectors. Protein concentrations ofall extracts were determined and each luciferase reading is normalizedfor protein concentration.

[0045] The 4.7 kb human MMP1 promoter driving luciferase was a gift ofConstance Brinckerhoff (Dartmouth Medical School) (36). The 804 bp humantype I collagen promoter driving luciferase was a gift from Russel Widom(Boston University). This construct, −804hCOL-LUC, contains a region ofthe type I alpha1 human collagen promoter from −804 to +114 bp. The 3TPlux construct, consisting of three AP1 sites and the plasminogenactivator inhibitor promoter (37,38), was a gift from Mikhail Panchenko(Boston University). The 4.5 kb protease nexin 1 promoter drivingluciferase (39) was a gift from Denis Guttridge (University of NorthCarolina, Chapel Hill).

[0046] Geldanamycin Treatment.

[0047] Geldanamycin was a generous gift of the National CancerInstitute. Geldanamycin was dissolved in dimethylsulfoxide (DMSO) andused at a final concentration of 2 μM except where indicated otherwise.DMSO without geldanamycin was added as the carrier control for allexperiments using geldanamycin.

[0048] Electrophoretic Mobility Shift Assays.

[0049] Nuclear extracts of 3T3 fibroblasts were obtained using standardmethods (40). Protein concentrations of extracts were determined usingthe BCA reagent (Pierce). Double stranded oligonucleotides correspondingto the 26 bp consensus Smad binding element (SBE) were generated (31).The probes were end-labeled using γ-³²P-ATP and T4 polynucleotidekinase. Electrophoretic mobility shift assays (EMSA) were performedusing 5 μg nuclear extract, 1 μl poly dI/dC (1 mg/ml), 0.1 ng of labeledprobe (in 1 μl), and buffer G to achieve a final volume of 10 μl. BufferG consists of 20 mM Hepes (pH 7.6), 100 mM KCl, 0.2 mM EDTA and 20%glycerol. Extracts and oligo were incubated 15 minutes at roomtemperature before adding loading buffer and running on a non-denaturinggel. Oct-1 duplex oligonucleotides were obtained from Santa CruzBiotechnology, Inc. Incubation of the extract and labeled SBEoligonucleotides was effectively competed with 100-fold molar excess ofcold nucleotide.

[0050] Double stranded oligonucleotides corresponding to the hsf1 (heatshock factor 1) binding site (5′-gcc tcg aat gtt cgc gaa gtt tcg and5′-cga aac ttc gcg aac att cga ggc) were generated and the EMSA wasperformed essentially as described in Goldenberg et al. (41).

[0051] Tail Vein Injection.

[0052] Tail veins of C57/Bl mice were injected with 10 μg of total DNAand TransIT lipid complex following the procedure of the TransITmanufacturer (Panvera, Madison Wis.). In brief, DNA was mixed with lipidcomplex, incubated at room temperature, and diluted with a volume ofdilution buffer equal to 1-tenth the animal weight (e.g., 3 mls for a 30g animal.) Mice were injected with 5 μg of the TGF-β-sensitive reporterp3TPlux and 5 μg of either a control vector (pBKRSV) or our Hsp90overexpression vector (pBKRSVHsp90). Mouse organs were harvested within24 hours. Liver tissue was dounce homogenized in 0.5 mls of reporterlysis buffer (Promega, Madison, Wis.). The extract was spun for 5minutes at 4 degrees to remove insoluble material. The supernatant wasassayed using the luciferase assay kit (Promega, Madison, Wis.): 5 μl ofsuspension was added to 100 μl of luciferase assay reagent.

[0053] The following examples are presented to illustrate the advantagesof the present invention and to assist one of ordinary skill in makingand using the same. These examples are not intended in any way otherwiseto limit the scope of the disclosure.

EXAMPLE I Hsp90α is Overexpressed in Scleroderma Fibroblasts

[0054] Polyadenylated mRNA was purified from healthy and diseased humandermal fibroblasts. Cells were obtained from both clinically lesionaland non-lesional skin of four systemic sclerosis patients and from 4healthy individuals. Radioactive labeled cDNA made from the mRNA wasused to probe commercially available filters containing 18,432 pairs ofrobotically spotted DNA samples (FIG. 1A). Filters were exposed tophosphoimage plates, and, initially, a visual inspection was performedto identify positions of clones that were differentially expressed inscleroderma fibroblasts. More than 30 clones were found that wereapparently overexpressed in scleroderma fibroblasts and more than 30that were expressed more highly in the healthy lines. Eighteen of theseclones were purchased and sequenced. Approximately half of these 18sequences did not match the putative sequences predicted by thesupplier. (However, when these clones were labeled and hybridized to theoriginal filter, 17 of them hybridized to the appropriate position.)

[0055]FIG. 1B shows a pattern of clones consistently overexpressed inscleroderma fibroblasts. The Figure shows the same region of filtersthat had been probed either with mRNA from healthy dermal fibroblasts(panels N-96-05, N-96-02 and N-92-04) or mRNA from lesional sclerodermafibroblasts (panels 96-02-A, 97-03-A and 97-02-A). The panels show twoclones (4 spots) which are not differentially expressed, labeled R, orreference clones. The panels also show three clones that are much morehighly expressed in the scleroderma fibroblasts. These three pairs ofspots are designated A, B and C. Clone A was sequenced and its sequencecorresponded to heat shock protein 90 alpha (Hsp90α).

[0056] Northern analysis of total RNA from cultured scleroderma andhealthy human dermal fibroblasts verified the differential expressionpredicted by the filter array results. FIG. 2 shows the northernresults. Lesional scleroderma lines were derived from biopsies of areasof patients' skin that contained phenotypically thickened tissues.Non-lesional scleroderma lines are derived from biopsies of areas ofpatients' skin that were phenotypically healthy. The northern analysisshows that Hsp90 is overexpressed in both lesional (L) and non-lesional(N) fibroblasts derived from biopsies of three independent sclerodermapatients (P1, P2, and P3). Only one of the 6 lines derived from biopsiesof healthy individuals (N1-N6) exhibited any Hsp90 signal.

[0057] Immunocytochemistry on cultured scleroderma and healthy humandermal cells showed that Hsp90 protein levels reflected the differentialexpression of the MRNA. FIG. 3 shows immunocytochemical staining ofthree healthy human dermal lines (panels A, B, C) and three lesionalscleroderma fibroblast lines (panels D, E, F) using a monoclonalanti-HSP90 antibody, AC88. The scleroderma lines showed intense stainingwhile the healthy lines showed little if any signal.

[0058] In addition to examining the expression of Hsp90 in sclerodermaand healthy human dermal fibroblasts, the expression and activity of thefactor thought to be of primary importance in the induction of heatshock proteins, heat shock factor 1 (HSF1) (41) was examined. Thiscytoplasmic protein responds to heat shock by forming a trimer andtranslocating to the nucleus, where it binds heat shock sensitiveelements to regulate transcription (42,43).

[0059] The amount of HSF1 DNA binding activity was determined byperforming EMSA assays on two different healthy dermal fibroblast linesand on two different scleroderma fibroblast lines. DNA binding sequenceswere synthesized based on previously published HSF1 DNA binding data(41). The results show that the basal level of HSF1 DNA binding activityin the nucleus of scleroderma cells is slightly higher than that inhealthy fibroblasts (FIG. 4, lanes 2-5). However, the level of HSF1 DNAbinding activity after a brief, 1.5 hour, heat shock at 42° is 144%higher in scleroderma cells (FIG. 4, lanes 7-10). In the brief heatshock period used, there was no significant increase in the HSF1 DNAbinding activity in the healthy cells. There was, however, a 48%increase in the level of HSF1 DNA binding activity in the sclerodermacells with heat shock. These results suggest that scleroderma cellsexhibit both a higher basal level of HSF1DNA binding activity and anincreased induction of this activity with heat shock.

EXAMPLE II Hsp90 Overexpression Induces Collagen and RepressesCollagenase

[0060] Collagen transcription activity was examined in a co-transfectionassay utilizing an 804 bp proximal region of the human type I collagenpromoter driving a luciferase reporter. Hsp90-α, Hsp90-β, Hsp70overexpression constructs or an empty vector control were co-transfectedwith the collagen promoter/reporter. FIG. 5 shows that in mouse NIH 3T3fibroblasts each of the three heat shock genes caused more than a 2-foldincrease in collagen promoter activity compared to the empty vectorwhich serves as the backbone for the overexpression constructs. The barsrepresent data from three transfections, each assayed in duplicate.

[0061] Net matrix accumulation in scleroderma might be caused either byincreased synthesis or decreased degradation of collagen. FIG. 6 showsthe results of an experiment designed to examine whether overexpressionof Hsp90 had reciprocal effects on collagen and collagenase (human MMP1)expression. Co-transfection of Hsp90-α with a 4.7 kb fragment of thehuman MMP1 promoter driving luciferase shows that Hsp90 caused an 8-folddecrease in collagenase promoter activity in 3T3 fibroblasts. Theseresults also indicate that Hsp90 overexpression does notindiscriminately activate promoters. Rather, Hsp90 both activates andrepresses different promoters in the same cell line.

EXAMPLE III Geldanamycin Inhibits Basal Collagen Synthesis andTGF-β-Induced Collagen Promoter Activation

[0062] The chaperone function of Hsp90 is dependent on the hydrolysis ofATP (44). Geldanamycin, a benzoquinone ansamycin antibiotic,specifically inhibits Hsp90 by binding to its ATP binding site (18).Geldanamycin was examined to determine whether its inhibition of Hsp90affected TGF-β activation of collagen transcription in mouse 3T3 andhuman dermal fibroblasts. Endogenous collagen message levels were usedas a measure of TGF-β signal transduction. The northern blots in FIG. 7shows the effect of geldanamycin on collagen message levels in theabsence or presence of TGF-β. The top panel (FIG. 7A) shows the effectin mouse 3T3 cells and the middle panel (FIG. 7B) in healthy humandermal fibroblasts. The left 4 lanes of this panel show, in the absenceof serum, that geldanamycin reduces the basal level of the collagentranscript. With TGF-β, the level of the endogenous collagen transcriptis increased approximately 6-fold. However, when geldanamycin and TGF-βare added together, the inhibitory effect of geldanamycin dominates.Thus, despite the 6-fold increase in collagen due to TGF-β, the additionof geldanamycin still reduces the collagen transcript level belowbaseline. The results in the presence of serum (right 4 lanes) aresimilar. The endogenous collagen transcript shows only a 2-fold increasewith TGF-β in the presence of serum, presumably because cells havealready acclimated to TGF-β or a related stimulus in the serum itself.However, geldanamycin alone or geldanamycin in the presence of TGF-βstill causes a reduction of collagen transcript to below baseline.

[0063] The middle panel (FIG. 7B) shows an analogous experiment usingprimary cultured healthy human dermal fibroblasts. Although theinduction of collagen in human cells by TGF-β is reduced, the resultsfollow the same trends as with mouse 3T3 fibroblasts. As before,geldanamycin alone reduces collagen synthesis and the induction ofcollagen by TGF-β is completely blocked by geldanamycin. This panel alsoshows a control hybridization of the blot using a labeled GAPDH probe.The GAPDH message level showed no variation, either with TGF-β or withgeldanamycin. As shown in the bottom panel (FIG. 7C), the same filterwas also stained with methylene blue to identify RNA beforehybridization. This staining revealed the 28S and 18S ribosomal bands asindicated, along with a faint smear which represents the rest of the RNAin each lane. As with the GAPDH, there was no indication of non-specificchanges in message level either with the addition of geldanamycin, TGF-βor both. These controls suggest that the effect of geldanamycin oncollagen message level is not a result of a general toxicity.

EXAMPLE IV Geldanamycin Blocks the TGF-β-Activated 3TP-lux Promoter andHas No Effect on the PN1 Promoter

[0064] Since geldanamycin blocked the activation of the collagenpromoter by TGF-β, the effect of geldanamycin on other promoterssensitive or insensitive to TGF-β was investigated. Mouse NIH 3T3 cellswere tranfected with the 3TPlux vector, which is known to be verysensitive to TGF-β, or a vector that contains a 4.5 kb portion of theprotease nexin 1 promoter driving luciferase. The PN1 promoter has notpreviously been shown to exhibit any sensitivity to TGF-β. However,previous studies had shown that PN1 is overexpressed in sclerodermafibroblasts and in scleroderma skin (10). Transfected cells were exposedto a 24 hour treatment of geldanamycin, TGF-β or both. Cells were thenharvested and assayed for luciferase activity.

[0065]FIG. 8 shows that the addition of geldanamycin slightly reducesthe level of expression of the TGF-β-sensitive promoter in the 3TPluxconstruct. As expected, TGF-β by itself resulted in a 3.5-fold inductionof the activity of this promoter. This induction, however, wascompletely blocked when geldanamycin and TGF-β were addedsimultaneously. The FIG. also shows that TGF-β has little effect on theexpression of the PN1 promoter. Geldanamycin neither reduces the basallevel of activity of this promoter nor reduces the activity of thispromoter in the presence of TGF-β. In fact, it seems that geldanamycinmay exert a small induction on the activity of the PN1 promoter.

[0066] These results, together with the data on collagen promoterinhibition in FIG. 7, suggest that the effect of geldanamycin onpromoter inhibition is somewhat specific for promoters that areactivated by TGF-β. Neither the PN1 nor the GAPDH promoters are blockedby the addition of geldanamycin. However, the TGF-β-sensitive promotersin the 3TPlux construct and the collagen gene are both subject toinhibition by geldanamycin. These facts led to a consideration ofwhether Hsp90 overexpression or geldanamycin inhibition of Hsp90 mayfunction on the TGF-β signaling pathway.

EXAMPLE V Hsp90 Induces the Expression of p3TP-lux When TransientlyExpressed In Vivo

[0067] To investigate further the role of Hsp90 in TGF-β signaltransduction, a novel method of tail vein injection was employed. Tailvein injection of either naked DNA or DNA complexed in lipophilicmoieties results in high levels of expression of the transgene ininternal organs, most prominently in the liver (45-47). Expressionlevels peak between 8 and 24 hours after injection, making this a rapidmethod to assess gene activity in vivo without resorting to the use of alarge number of mice as required in traditional transgenic studies.

[0068] While previous studies examined the expression of a singlereporter, this study expanded that work by co-injecting both apromoter-reporter and an overexpression plasmid. In this study, 5 μg ofTGF-β-sensitive reporter plasmid (p3TP-lux) was co-injected with 5 μg ofeither an empty control vector (pBKRSV) or a plasmid that overexpressesHsp90-α (pBKRSVHsp90). Generally, 3 ml of a DNA-lipophilic agent mixturewere injected as detailed in Experimental Procedures.

[0069] In pilot studies, luciferase activities were measured in extractsfrom heart, lung, thymus, liver, kidney and spleen. There was very lowexpression in all organs except liver. In subsequent experiments,luciferase activity was measured only in extracts of liver tissue, andalways within 16-24 hours after the injection. FIG. 9 shows that the3TPlux reporter was significantly expressed only when Hsp90 wasco-expressed with the reporter. Hsp90 expression induced the reporter byat least five-fold in all cases. In some cases, Hsp90 induced thereporter several hundred fold. These results complement the earlierdata, which demonstrate that inhibiting Hsp90 blocks 3TPlux expression(FIG. 8) and blocks the TGF-β response of the endogenous collagen gene(FIG. 7). Further studies should reveal whether the overexpression ofHsp90 in the liver also induces the expression of endogenous transcriptsthat are activated by TGF-β.

EXAMPLE VI Geldanamycin Inhibits Activation of a Smad-ControlledPromoter and Decreases Smad DNA Binding

[0070] To examine whether the effect of geldanamycin was exerted onSmads, the signal transduction protein for TGF-β, a Smad controlledreporter plasmid, was constructed identically to a previously reportedvector (31). Smad 3 and Smad 4 bind to specific sequences which werecloned into the pGL3 luciferase reporter vector, which otherwise had aminimal promoter. The Smad-controlled reporter or the pGL3pv controlplasmid were transfected into 3T3 fibroblasts in the presence or absenceof increasing concentrations of geldanamycin. FIG. 10 demonstrates thatSmad binding sequences cause a dramatic increase in reportertranscription even in the absence of exogenously supplied TGF-β. Thissuggests that 3T3 fibroblasts have a considerable baseline level ofactive Smad, a finding that has also been reported in melanoma cells(48). Geldanamycin reduced Smad-dependent transcription by two-thirds.This suggests that geldanamycin prevents the Smad signaling by eitherpreventing the activation of Smad or preventing translocation of activeSmad to the nucleus.

[0071] Electrophoretic mobility shift assays were performed to examinewhether geldanamycin reduced the level of Smad DNA-binding activity innuclear extracts. As before, cells were also subjected to TGF-β or TGF-βand geldanamycin together. In this experiment (FIG. 11A), cells werepretreated for 1 hour with geldanamycin and then given a brief,15-minute treatment with TGF-β prior to harvesting nuclear extracts forbinding. The level of Smad bound to its Smad binding element wasmodestly reduced relative to control by the 1 hour treatment with 20 μMgeldanamycin (compare the first and second lanes). TGF-β, as expected,caused a modest increase in nuclear Smad after a 15 minute treatment.This level of increase, however, was reduced to below the control by thetreatment with geldanamycin before the addition of TGF-β (fourth lane).By contrast, the level of binding of Smad from 3T3 nuclear extracts tothe Oct1 binding site (FIG. 11B) was not significantly changed in any ofthe concentrations of geldanamycin during a 24 hour treatment.

[0072] Use

[0073] The results reported here show that compounds capable ofinterfering with the activity of a collagen promotor are good candidatesfor prophylaxis or treatment of scleroderma and other fibrogenicdiseases or disorders. The Hsp90-α chaperone function inhibitorsdescribed above have been shown to be strong inhibitors of TGF-β-inducedexpression of reporters under the control of the collagen promoter.Therefore, they will be very useful in therapeutic compositions andmethods for treating patients with, or believed to be at risk ofacquiring, fibrogenic disorders. The therapeutic compositions may beadministered topically, orally, or parenterally, (e.g., intranasally,subcutaneously, intramuscularly, intravenously, or intra-arterially) byroutine methods in pharmaceutically acceptable inert carrier substances.For example, the therapeutic compositions of the invention may beadministered locally by direct application in a carrier vehicle, byon-site delivery using micelles, gels or liposomes, or in a sustainedrelease formulation using a biodegradable biocompatible polymer. Thetherapeutic agents can be administered, e.g., locally, in a dosage of0.05 μg/kg/day to 10 μg/kg/day (and preferably 0.25 μg/kg/day to 2.5μg/kg/day) for a total of, e.g., 50 μg/day for a 70 kg human patient.Optimal dosage and modes of administration can readily be determined byconventional protocols.

[0074] Preferred inhibitors according to the invention include knowninhibitors of Hsp90-α function, and in particular, geldanamycin. Sincegeldanamycin is a small organic molecule, it is readily amenable to anumber of modifications, as is well known to those of ordinary skill inthe art. Such modifications can include, but not be limited to,additions of carbonyl, amine, hydroxyl and other groups to the reactivesites already on geldanamycin, including the oxygen and nitrogenpositions. The purpose of such modifications is, e.g., to enhance druguptake in human and animal systems as well as to enhance theeffectiveness of the drug in blocking the TGF-β signaling pathway and inblocking the production of collagen and other matrix components.

[0075] Three systems that can be used to measure the effectiveness ofsuch modifications or, in general, to screen candidate inhibitors,include:

[0076] (1) A mouse fibroblast-derived cell line stably transfected witha TGF-β-responsive promoter driving a luciferase reporter (p3TP-lux).Such a cell line gives a 10-30-fold increase in luciferase activity inresponse to TGF-β. This increase is known to be substantially blocked bygeldanamycin, and the effectiveness of a candidate inhibitor can becompared to the activity of geldanamycin as a positive control.

[0077] (2) Mouse and human cell lines stably transfected with a collagenpromoter driving luciferase and/or green fluorescent protein reporters.These cell lines can be used to demonstrate the effect of geldanamycin,its derivatives and related candidate inhibitors of Hsp90 in blockingthe TGF-β-induced expression of reporters under the control of thecollagen promoter.

[0078] (3) Northern analysis of endogenous collagen message can be canbe carried out on healthy human dermal fibroblasts to measure the effectof geldanamycin, derivatives and related inhibitors on the transcriptionof the endogenous collagen message.

[0079] While the present invention has been described in conjunctionwith a preferred embodiment, one of ordinary skill, after reading theforegoing specification, will be able to effect various changes,substitutions of equivalents, and other alterations to the compositionsand methods set forth herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by thedefinitions contained in the appended claims and equivalents thereof.

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What is claimed is:
 1. A method of prophylaxis or treatment of afibrogenic disorder in a patient, said method comprising the steps of:providing a patient suffering from or believed to be at risk ofsuffering from a fibrogenic disorder; and administering to said patientan effective amount of a therapeutic composition comprising an inhibitorof collagen promoter activity in a pharmaceutically acceptable inertcarrier vehicle, wherein said amount of said composition is effective inpreventing or treating said fibrogenic disorder.
 2. The method of claim1, wherein, in said administering step, said composition is administeredlocally.
 3. The method of claim 2, wherein, in said administering step,said composition is administered topically.
 4. The method of claim 1,wherein said inhibitor is effective in blocking TGF-β signaltransduction.
 5. The method of claim 1, wherein said inhibitorinterferes with heat shock protein 90 chaperone function.
 6. The methodof claim 1, wherein said inhibitor is capable of binding to an ATPbinding site of heat shock protein
 90. 7. The method of claim 1,wherein, in said administering step, said composition is administered ata concentration that is insufficient to detectably affect steroidhormone receptor activity.
 8. The method of claim 1, wherein, in saidadministering step, said composition is administered locally at aconcentration of 0.25 μg/kg/day to 2.5 μg/kg/day.
 9. The method of claim1, wherein said composition comprises a compound selected from the groupconsisting of geldanamycin, macbecin I and II, herbimycin, radcicol andnovobiocin.
 10. The method of claim 9, wherein said compositioncomprises geldanamycin.
 11. The method of claim 1, wherein saidfibrogenic disorder is selected from the group consisting ofscleroderma, polymyositis, systemic lupus erythematosis, rheumatoidarthritis, keloid formation, interstitial nephritis and pulmonaryfibrosis.
 12. The method of claim 11, wherein said fibrogenic disorderis selected from the group consisting of scleroderma, keloid formationand liver cirrhosis.
 13. A therapeutic composition comprising aninhibitor of heat shock protein 90 alpha chaperone function; and apharmaceutically acceptable inert carrier vehicle for topicalapplication.
 14. The composition of claim 13, wherein said inhibitor isselected from the group consisting of geldanamycin, macbecin I and II,herbimycin, radcicol and novobiocin.
 15. The composition of claim 14,wherein said inhibitor is geldanamycin.
 16. An article of manufacturecomprising a packaging material; and a therapeutic composition containedwithin said packaging material, wherein said therapeutic compositioncomprises an inhibitor of a collagen promoter in a pharmaceuticallyacceptable inert carrier vehicle, and wherein said packaging materialalso comprises a label with instructions for use, said instructionsindicating that said therapeutic composition can be used for prophylaxisor treatment of a fibrogenic disorder.
 17. The article of manufacture ofclaim 16, wherein said composition comprises a compound selected fromthe group consisting of geldanamycin, macbecin I and II, herbimycin,radcicol and novobiocin.
 18. The article of manufacture of claim 17,wherein said composition comprises geldanamycin.
 19. The article ofmanufacture of claim 16, wherein said fibrogenic disorder is selectedfrom the group consisting of scleroderma, polymyositis, systemic lupuserythematosis, rheumatoid arthritis, keloid formation, interstitialnephritis and pulmonary fibrosis.
 20. The article of manufaacture ofclaim 19, wherein said fibrogenic disorder is selected from the groupconsisting of scleroderma, keloid formation and liver cirrhosis.